Team:Calgary/27 July 2009

From 2009.igem.org

(Difference between revisions)
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<html>
<html>
Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers
Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers
-
 
+
<br>
Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.
Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.
<br>
<br>
Line 416: Line 416:
Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3
Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3
-
 
+
<br>
 +
<br>
PCR conditions
PCR conditions
 +
<html>
 +
<table x:str border=0 cellpadding=0 cellspacing=0 width=314 style='border-collapse:
 +
collapse;table-layout:fixed;width:235pt'>
 +
<col width=92 style='mso-width-source:userset;mso-width-alt:3364;width:69pt'>
 +
<col width=91 style='mso-width-source:userset;mso-width-alt:3328;width:68pt'>
 +
<col width=131 style='mso-width-source:userset;mso-width-alt:4790;width:98pt'>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl24 width=92 style='height:16.5pt;width:69pt'><span
 +
  lang=DA style='mso-ansi-language:DA'># of cycles</span></td>
 +
  <td class=xl25 width=91 style='width:68pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>Temp (ºC)</span></td>
 +
  <td class=xl25 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>Time</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=92 style='height:16.5pt;width:69pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>1</span></td>
 +
  <td class=xl27 width=91 style='width:68pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>94</span></td>
 +
  <td class=xl27 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>6 min</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
-
Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.
+
  <td rowspan=3 height=66 class=xl28 width=92 style='border-bottom:1.0pt solid black;
 +
  height:49.5pt;border-top:none;width:69pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>36</span></td>
 +
  <td class=xl27 width=91 style='width:68pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>94</span></td>
 +
  <td class=xl27 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>30sec</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl27 width=91 style='height:16.5pt;width:68pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>55</span></td>
 +
  <td class=xl27 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>45sec</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl27 width=91 style='height:16.5pt;width:68pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>72</span></td>
 +
  <td class=xl27 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>6min 20sec</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=92 style='height:16.5pt;width:69pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>1</span></td>
 +
  <td class=xl27 width=91 style='width:68pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>72</span></td>
 +
  <td class=xl27 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>10min</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=92 style='height:16.5pt;width:69pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>&nbsp;</span></td>
 +
  <td class=xl27 width=91 style='width:68pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>4</span></td>
 +
  <td class=xl27 width=131 style='width:98pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>hold</span></td>
 +
</tr>
 +
<![if supportMisalignedColumns]>
 +
<tr height=0 style='display:none'>
 +
  <td width=92 style='width:69pt'></td>
 +
  <td width=91 style='width:68pt'></td>
 +
  <td width=131 style='width:98pt'></td>
 +
</tr>
 +
<![endif]>
 +
</table>
 +
</html>
 +
Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.
 +
<br>
 +
<br>
 +
<br>
 +
Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers
 +
<br>
 +
Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)
 +
<br>
 +
Protocol
 +
<br>
 +
<html>
 +
<table x:str border=0 cellpadding=0 cellspacing=0 width=667 style='border-collapse:
 +
collapse;table-layout:fixed;width:501pt'>
 +
<col width=198 style='mso-width-source:userset;mso-width-alt:7241;width:149pt'>
 +
<col width=205 style='mso-width-source:userset;mso-width-alt:7497;width:154pt'>
 +
<col width=264 style='mso-width-source:userset;mso-width-alt:9654;width:198pt'>
 +
<tr height=22 style='height:16.5pt;mso-yfti-firstrow:yes;mso-yfti-irow:0'>
 +
  <td height=22 class=xl24 width=198 style='height:16.5pt;width:149pt'>&nbsp;</td>
 +
  <td class=xl25 width=205 style='width:154pt'>MM1 (8x) (&#956;L)</td>
 +
  <td class=xl25 width=264 style='width:198pt'>MM2 (6x) (&#956;L)</td>
 +
</tr>
 +
<tr height=26 style='height:19.5pt'>
 +
  <td height=26 class=xl26 width=198 style='height:19.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>10X PCR buffer minus MgCl<font
 +
  class="font6"><sub>2</sub></font></span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>40</span></td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>40</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>10mM dNTPs</span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>8</span></td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>8</span></td>
 +
</tr>
 +
<tr height=26 style='height:19.5pt'>
 +
  <td height=26 class=xl26 width=198 style='height:19.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>50mM MgCl<font class="font6"><sub>2</sub></font></span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>12</span></td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>12</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>F primer</span></td>
 +
  <td class=xl27 width=205 style='width:154pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>8 (LuxPQ F)</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>8 (BBK CP F)</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>R primer</span></td>
 +
  <td class=xl27 width=205 style='width:154pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>8 (LuxPQ R)</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>8 (BBK CP R)</span></td>
 +
</tr>
 +
<tr height=26 style='height:19.5pt'>
 +
  <td height=26 class=xl26 width=198 style='height:19.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>ddH<font class="font6"><sub>2</sub></font><font
 +
  class="font5">O</font></span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>322.4</span></td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>322.4</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>pTaq</span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>1.6</span></td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>1.6</span></td>
 +
</tr>
 +
<![if supportMisalignedColumns]>
 +
<tr height=0 style='display:none'>
 +
  <td width=198 style='width:149pt'></td>
 +
  <td width=205 style='width:154pt'></td>
 +
  <td width=264 style='width:198pt'></td>
 +
</tr>
 +
<![endif]>
 +
</table>
 +
</html>
 +
<br>
 +
Positive control = ∆LuxPQ in psB1AK3
 +
<br>
 +
<br>
 +
PCR conditions
 +
<br>
 +
<hmtl>
 +
<table x:str border=0 cellpadding=0 cellspacing=0 width=667 style='border-collapse:
 +
collapse;table-layout:fixed;width:501pt'>
 +
<col width=198 style='mso-width-source:userset;mso-width-alt:7241;width:149pt'>
 +
<col width=205 style='mso-width-source:userset;mso-width-alt:7497;width:154pt'>
 +
<col width=264 style='mso-width-source:userset;mso-width-alt:9654;width:198pt'>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl24 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'># of cycles</span></td>
 +
  <td class=xl25 width=205 style='width:154pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>Temp (ºC)</span></td>
 +
  <td class=xl25 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>Time</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>1</span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>94</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>6 min</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td rowspan=3 height=66 class=xl28 width=198 style='border-bottom:1.0pt solid black;
 +
  height:49.5pt;border-top:none;width:149pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>36</span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>94</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>30sec</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl27 width=205 style='height:16.5pt;width:154pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>55</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>45sec</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl27 width=205 style='height:16.5pt;width:154pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>72</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>6min 20sec</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt' x:num><span
 +
  lang=DA style='mso-ansi-language:DA'>1</span></td>
-
Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers
+
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>72</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>10min</span></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>&nbsp;</span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>4</span></td>
 +
  <td class=xl27 width=264 style='width:198pt'><span lang=DA style='mso-ansi-language:
 +
  DA'>hold</span></td>
-
Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)
+
</tr>
 +
<tr height=22 style='height:16.5pt'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'><span
 +
  lang=DA style='mso-ansi-language:DA'>pTaq</span></td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>1.6</span></td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num><span lang=DA
 +
  style='mso-ansi-language:DA'>1.6</span></td>
 +
</tr>
 +
<![if supportMisalignedColumns]>
-
Protocol
+
<tr height=0 style='display:none'>
 +
  <td width=198 style='width:149pt'></td>
 +
  <td width=205 style='width:154pt'></td>
 +
  <td width=264 style='width:198pt'></td>
 +
</tr>
 +
<![endif]>
 +
</table>
 +
</html>
 +
<br>
 +
<br>
 +
Image
 +
<br>
 +
Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)
 +
<br>
 +
Purpose: isolate and measure concentrations of pure plasmids.
 +
<br>
 +
<html>
 +
<table x:str border=0 cellpadding=0 cellspacing=0 width=731 style='border-collapse:
 +
collapse;table-layout:fixed;width:549pt'>
 +
<col width=198 style='mso-width-source:userset;mso-width-alt:7241;width:149pt'>
 +
<col width=205 style='mso-width-source:userset;mso-width-alt:7497;width:154pt'>
 +
<col width=264 style='mso-width-source:userset;mso-width-alt:9654;width:198pt'>
 +
<col width=64 style='width:48pt'>
 +
<tr height=43 style='height:32.25pt;mso-yfti-firstrow:yes;mso-yfti-irow:0'>
 +
  <td height=43 class=xl24 width=198 style='height:32.25pt;width:149pt'>DNA</td>
 +
  <td class=xl25 width=205 style='width:154pt'>260/280</td>
-
Positive control = ∆LuxPQ in psB1AK3
+
  <td class=xl25 width=264 style='width:198pt'>260/230</td>
 +
  <td class=xl25 width=64 style='width:48pt'>Conc. [ng/&#956;L]</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:1'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>&#8710;LuxPQ
 +
  in AC3 C1</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.83</td>
-
PCR conditions
+
  <td class=xl27 width=264 style='width:198pt' x:num>2.23</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>146.8</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:2'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>&#8710;LuxPQ
 +
  in AC3 C2</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.81</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>2.19</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>112.6</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:3'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>&#8710;LuxPQ
 +
  in AC3 C3</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.8</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>2.27</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>145.8</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:4'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>&#8710;LuxPQ
 +
  in AC3 C4</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.78</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>2.09</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>96.6</td>
 +
</tr>
-
Result: Of the 6 colonies, 5 had bands of the correct size (~3.9kb).
+
<tr height=22 style='height:16.5pt;mso-yfti-irow:5'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>&#8710;LuxPQ
 +
  in AC3 C5</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.81</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>2.25</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>115.3</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:6'>
-
   
+
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>&#8710;LuxPQ
 +
  in AC3 C6</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.79</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>2.17</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>44.8</td>
 +
  </tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:7'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>PQ-B-R-OU-B
 +
  in AC3 C1</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.76</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>1.86</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>29.4</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:8'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>PQ-B-R-OU-B
 +
  in AC3 C2</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.59</td>
-
Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)
+
  <td class=xl27 width=264 style='width:198pt' x:num>1.44</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>20.1</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:9'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>PQ-B-R-OU-B
 +
  in AC3 C3</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.7</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>2.27</td>
-
Purpose: isolate and measure concentrations of pure plasmids.
+
  <td class=xl27 width=64 style='width:48pt' x:num>16.3</td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:10;mso-yfti-lastrow:yes'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>PQ-B-R-OU-B
 +
  in AC3 C4</td>
 +
  <td class=xl27 width=205 style='width:154pt' x:num>1.71</td>
 +
  <td class=xl27 width=264 style='width:198pt' x:num>1.79</td>
 +
  <td class=xl27 width=64 style='width:48pt' x:num>17</td>
 +
</tr>
 +
<![if supportMisalignedColumns]>
 +
<tr height=0 style='display:none'>
 +
  <td width=198 style='width:149pt'></td>
 +
  <td width=205 style='width:154pt'></td>
 +
  <td width=264 style='width:198pt'></td>
 +
  <td width=64 style='width:48pt'></td>
 +
</tr>
 +
<![endif]>
 +
</table>
 +
</html>
 +
<br>
 +
<br>
 +
<br>
 +
Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
 +
<br>
 +
<br>
 +
Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
 +
<br>
 +
Protocol
 +
<br>
 +
Recipient
 +
<html>
 +
<table x:str border=0 cellpadding=0 cellspacing=0 width=731 style='border-collapse:
 +
collapse;table-layout:fixed;width:549pt'>
 +
<col width=198 style='mso-width-source:userset;mso-width-alt:7241;width:149pt'>
 +
<col width=205 style='mso-width-source:userset;mso-width-alt:7497;width:154pt'>
 +
<col width=264 style='mso-width-source:userset;mso-width-alt:9654;width:198pt'>
 +
<col width=64 style='width:48pt'>
 +
<tr height=22 style='height:16.5pt;mso-yfti-firstrow:yes;mso-yfti-irow:0'>
 +
  <td height=22 class=xl25 width=198 style='height:16.5pt;width:149pt'>Recipient
 +
  1</td>
 +
  <td class=xl26 width=205 style='width:154pt'>Recipient 2</td>
 +
  <td class=xl24 width=264 style='width:198pt'></td>
 +
  <td class=xl24 width=64 style='width:48pt'></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:1'>
 +
  <td height=22 class=xl27 width=198 style='height:16.5pt;width:149pt'>2&#956;L
 +
  REact 4</td>
 +
  <td class=xl28 width=205 style='width:154pt'>2&#956;L REact 4</td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:2'>
 +
  <td height=22 class=xl27 width=198 style='height:16.5pt;width:149pt'>0.75
 +
  &#956;L SpeI</td>
 +
  <td class=xl28 width=205 style='width:154pt'>0.75 &#956;L SpeI</td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:3'>
 +
  <td height=22 class=xl27 width=198 style='height:16.5pt;width:149pt'>0.75
 +
  &#956;L PstI</td>
 +
  <td class=xl28 width=205 style='width:154pt'>0.75 &#956;L PstI</td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=43 style='height:32.25pt;mso-yfti-irow:4'>
 +
  <td height=43 class=xl27 width=198 style='height:32.25pt;width:149pt'>2
 +
  &#956;L of &#8710;LuxPQ in AK3 C2 [112.6ng/&#956;L]</td>
 +
  <td class=xl28 width=205 style='width:154pt'>2 &#956;L of &#8710;LuxPQ in AK3
 +
  C5 [115.3ng/&#956;L]</td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:5;mso-yfti-lastrow:yes'>
 +
  <td height=22 class=xl27 width=198 style='height:16.5pt;width:149pt'>14.5
 +
  &#956;L ddH2O</td>
 +
  <td class=xl28 width=205 style='width:154pt'>14.5 &#956;L ddH2O</td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<![if supportMisalignedColumns]>
 +
<tr height=0 style='display:none'>
 +
  <td width=198 style='width:149pt'></td>
 +
  <td width=205 style='width:154pt'></td>
 +
  <td width=264 style='width:198pt'></td>
 +
  <td width=64 style='width:48pt'></td>
 +
</tr>
 +
<![endif]>
 +
</table>
 +
</html>
 +
<br>
 +
<br>
 +
Insert
 +
<html>
 +
<table x:str border=0 cellpadding=0 cellspacing=0 width=731 style='border-collapse:
 +
collapse;table-layout:fixed;width:549pt'>
 +
<col width=198 style='mso-width-source:userset;mso-width-alt:7241;width:149pt'>
 +
<col width=205 style='mso-width-source:userset;mso-width-alt:7497;width:154pt'>
 +
<col width=264 style='mso-width-source:userset;mso-width-alt:9654;width:198pt'>
 +
<col width=64 style='width:48pt'>
 +
<tr height=22 style='height:16.5pt;mso-yfti-firstrow:yes;mso-yfti-irow:0'>
 +
  <td height=22 class=xl25 width=198 style='height:16.5pt;width:149pt'>2&#956;L
 +
  REact 2</td>
 +
  <td class=xl27 width=205 style='width:154pt'></td>
 +
  <td class=xl24 width=264 style='width:198pt'></td>
 +
  <td class=xl24 width=64 style='width:48pt'></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:1'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>0.75
 +
  &#956;L XbaI</td>
 +
  <td class=xl27></td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
-
Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
+
<tr height=22 style='height:16.5pt;mso-yfti-irow:2'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>0.75
 +
  &#956;L PstI</td>
 +
  <td class=xl27></td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=64 style='height:48.0pt;mso-yfti-irow:3'>
 +
  <td height=64 class=xl26 width=198 style='height:48.0pt;width:149pt'>2
 +
  &#956;L of LuxPQ-B0015-R0040-LuxOU-B0015 in AK3 C1 [156.3ng/&#956;L]</td>
-
Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
+
  <td class=xl27></td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=22 style='height:16.5pt;mso-yfti-irow:4;mso-yfti-lastrow:yes'>
 +
  <td height=22 class=xl26 width=198 style='height:16.5pt;width:149pt'>12.5
 +
  &#956;L ddH2O</td>
 +
  <td class=xl27></td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
-
Protocol
+
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=2 class=xl27 style='height:15.75pt;mso-ignore:colspan'></td>
 +
  <td colspan=2 class=xl24 style='mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
-
Recipient
+
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<tr height=21 style='height:15.75pt'>
 +
  <td height=21 colspan=4 class=xl24 style='height:15.75pt;mso-ignore:colspan'></td>
 +
</tr>
 +
<![if supportMisalignedColumns]>
 +
<tr height=0 style='display:none'>
 +
  <td width=198 style='width:149pt'></td>
 +
  <td width=205 style='width:154pt'></td>
 +
  <td width=264 style='width:198pt'></td>
 +
  <td width=64 style='width:48pt'></td>
 +
</tr>
 +
<![endif]>
-
Insert
+
</table>
-
 
+
</html>
 +
<br>
 +
<br>
Put in the incubator at 37ºC overnight.
Put in the incubator at 37ºC overnight.

Revision as of 16:29, 29 July 2009

University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

July
MTWTFSS
    [http://2009.igem.org/Team:Calgary/1_July_2009 1] [http://2009.igem.org/Team:Calgary/2_July_2009 2] [http://2009.igem.org/Team:Calgary/3_July_2009 3] [http://2009.igem.org/Team:Calgary/4_July_2009 4] [http://2009.igem.org/Team:Calgary/5_July_2009 5]
[http://2009.igem.org/Team:Calgary/6_July_2009 6] [http://2009.igem.org/Team:Calgary/7_July_2009 7] [http://2009.igem.org/Team:Calgary/8_July_2009 8] [http://2009.igem.org/Team:Calgary/9_July_2009 9] [http://2009.igem.org/Team:Calgary/10_July_2009 10] [http://2009.igem.org/Team:Calgary/11_July_2009 11] [http://2009.igem.org/Team:Calgary/12_July_2009 12]
[http://2009.igem.org/Team:Calgary/13_July_2009 13] [http://2009.igem.org/Team:Calgary/14_July_2009 14] [http://2009.igem.org/Team:Calgary/15_July_2009 15] [http://2009.igem.org/Team:Calgary/16_July_2009 16] [http://2009.igem.org/Team:Calgary/17_July_2009 17] [http://2009.igem.org/Team:Calgary/18_July_2009 18] [http://2009.igem.org/Team:Calgary/19_July_2009 19]
[http://2009.igem.org/Team:Calgary/20_July_2009 20] [http://2009.igem.org/Team:Calgary/21_July_2009 21] [http://2009.igem.org/Team:Calgary/22_July_2009 22] [http://2009.igem.org/Team:Calgary/23_July_2009 23] [http://2009.igem.org/Team:Calgary/24_July_2009 24] [http://2009.igem.org/Team:Calgary/25_July_2009 25] [http://2009.igem.org/Team:Calgary/26_July_2009 26]
[http://2009.igem.org/Team:Calgary/27_July_2009 27] [http://2009.igem.org/Team:Calgary/28_July_2009 28] [http://2009.igem.org/Team:Calgary/29_July_2009 29] [http://2009.igem.org/Team:Calgary/30_July_2009 30] [http://2009.igem.org/Team:Calgary/31_July_2009 31]


NOTEBOOK PAGE INDEX
Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki



CALENDAR

May
MTWTFSS
        [http://2009.igem.org/Team:Calgary/1_May_2009 1] [http://2009.igem.org/Team:Calgary/2_May_2009 2] [http://2009.igem.org/Team:Calgary/3_May_2009 3]
[http://2009.igem.org/Team:Calgary/4_May_2009 4] [http://2009.igem.org/Team:Calgary/5_May_2009 5] [http://2009.igem.org/Team:Calgary/6_May_2009 6] [http://2009.igem.org/Team:Calgary/7_May_2009 7] [http://2009.igem.org/Team:Calgary/8_May_2009 8] [http://2009.igem.org/Team:Calgary/9_May_2009 9] [http://2009.igem.org/Team:Calgary/10_May_2009 10]
[http://2009.igem.org/Team:Calgary/11_May_2009 11] [http://2009.igem.org/Team:Calgary/12_May_2009 12] [http://2009.igem.org/Team:Calgary/13_May_2009 13] [http://2009.igem.org/Team:Calgary/14_May_2009 14] [http://2009.igem.org/Team:Calgary/15_May_2009 15] [http://2009.igem.org/Team:Calgary/16_May_2009 16] [http://2009.igem.org/Team:Calgary/17_May_2009 17]
[http://2009.igem.org/Team:Calgary/18_May_2009 18] [http://2009.igem.org/Team:Calgary/19_May_2009 19] [http://2009.igem.org/Team:Calgary/20_May_2009 20] [http://2009.igem.org/Team:Calgary/21_May_2009 21] [http://2009.igem.org/Team:Calgary/22_May_2009 22] [http://2009.igem.org/Team:Calgary/23_May_2009 23] [http://2009.igem.org/Team:Calgary/24_May_2009 24]
[http://2009.igem.org/Team:Calgary/25_May_2009 25] [http://2009.igem.org/Team:Calgary/26_May_2009 26] [http://2009.igem.org/Team:Calgary/27_May_2009 27] [http://2009.igem.org/Team:Calgary/28_May_2009 28] [http://2009.igem.org/Team:Calgary/29_May_2009 29] [http://2009.igem.org/Team:Calgary/30_May_2009 30] [http://2009.igem.org/Team:Calgary/31_May_2009 31]


June
MTWTFSS
[http://2009.igem.org/Team:Calgary/1_June_2009 1] [http://2009.igem.org/Team:Calgary/2_June_2009 2] [http://2009.igem.org/Team:Calgary/3_June_2009 3] [http://2009.igem.org/Team:Calgary/4_June_2009 4] [http://2009.igem.org/Team:Calgary/5_June_2009 5] [http://2009.igem.org/Team:Calgary/6_June_2009 6] [http://2009.igem.org/Team:Calgary/7_June_2009 7]
[http://2009.igem.org/Team:Calgary/8_June_2009 8] [http://2009.igem.org/Team:Calgary/9_June_2009 9] [http://2009.igem.org/Team:Calgary/10_June_2009 10] [http://2009.igem.org/Team:Calgary/11_June_2009 11] [http://2009.igem.org/Team:Calgary/12_June_2009 12] [http://2009.igem.org/Team:Calgary/13_June_2009 13] [http://2009.igem.org/Team:Calgary/14_June_2009 14]
[http://2009.igem.org/Team:Calgary/15_June_2009 15] [http://2009.igem.org/Team:Calgary/16_June_2009 16] [http://2009.igem.org/Team:Calgary/17_June_2009 17] [http://2009.igem.org/Team:Calgary/18_June_2009 18] [http://2009.igem.org/Team:Calgary/19_June_2009 19] [http://2009.igem.org/Team:Calgary/20_June_2009 20] [http://2009.igem.org/Team:Calgary/21_June_2009 21]
[http://2009.igem.org/Team:Calgary/22_June_2009 22] [http://2009.igem.org/Team:Calgary/23_June_2009 23] [http://2009.igem.org/Team:Calgary/24_June_2009 24] [http://2009.igem.org/Team:Calgary/25_June_2009 25] [http://2009.igem.org/Team:Calgary/26_June_2009 26] [http://2009.igem.org/Team:Calgary/27_June_2009 27] [http://2009.igem.org/Team:Calgary/28_June_2009 28]
[http://2009.igem.org/Team:Calgary/29_June_2009 29] [http://2009.igem.org/Team:Calgary/30_June_2009 30]


July
MTWTFSS
    [http://2009.igem.org/Team:Calgary/1_July_2009 1] [http://2009.igem.org/Team:Calgary/2_July_2009 2] [http://2009.igem.org/Team:Calgary/3_July_2009 3] [http://2009.igem.org/Team:Calgary/4_July_2009 4] [http://2009.igem.org/Team:Calgary/5_July_2009 5]
[http://2009.igem.org/Team:Calgary/6_July_2009 6] [http://2009.igem.org/Team:Calgary/7_July_2009 7] [http://2009.igem.org/Team:Calgary/8_July_2009 8] [http://2009.igem.org/Team:Calgary/9_July_2009 9] [http://2009.igem.org/Team:Calgary/10_July_2009 10] [http://2009.igem.org/Team:Calgary/11_July_2009 11] [http://2009.igem.org/Team:Calgary/12_July_2009 12]
[http://2009.igem.org/Team:Calgary/13_July_2009 13] [http://2009.igem.org/Team:Calgary/14_July_2009 14] [http://2009.igem.org/Team:Calgary/15_July_2009 15] [http://2009.igem.org/Team:Calgary/16_July_2009 16] [http://2009.igem.org/Team:Calgary/17_July_2009 17] [http://2009.igem.org/Team:Calgary/18_July_2009 18] [http://2009.igem.org/Team:Calgary/19_July_2009 19]
[http://2009.igem.org/Team:Calgary/20_July_2009 20] [http://2009.igem.org/Team:Calgary/21_July_2009 21] [http://2009.igem.org/Team:Calgary/22_July_2009 22] [http://2009.igem.org/Team:Calgary/23_July_2009 23] [http://2009.igem.org/Team:Calgary/24_July_2009 24] [http://2009.igem.org/Team:Calgary/25_July_2009 25] [http://2009.igem.org/Team:Calgary/26_July_2009 26]
[http://2009.igem.org/Team:Calgary/27_July_2009 27] [http://2009.igem.org/Team:Calgary/28_July_2009 28] [http://2009.igem.org/Team:Calgary/29_July_2009 29] [http://2009.igem.org/Team:Calgary/30_July_2009 30] [http://2009.igem.org/Team:Calgary/31_July_2009 31]


August
MTWTFSS
          [http://2009.igem.org/Team:Calgary/1_August_2009 1] [http://2009.igem.org/Team:Calgary/2_August_2009 2]
[http://2009.igem.org/Team:Calgary/3_August_2009 3] [http://2009.igem.org/Team:Calgary/4_August_2009 4] [http://2009.igem.org/Team:Calgary/5_August_2009 5] [http://2009.igem.org/Team:Calgary/6_August_2009 6] [http://2009.igem.org/Team:Calgary/7_August_2009 7] [http://2009.igem.org/Team:Calgary/8_August_2009 8] [http://2009.igem.org/Team:Calgary/9_August_2009 9]
[http://2009.igem.org/Team:Calgary/10_August_2009 10] [http://2009.igem.org/Team:Calgary/11_August_2009 11] [http://2009.igem.org/Team:Calgary/12_August_2009 12] [http://2009.igem.org/Team:Calgary/13_August_2009 13] [http://2009.igem.org/Team:Calgary/14_August_2009 14] [http://2009.igem.org/Team:Calgary/15_August_2009 15] [http://2009.igem.org/Team:Calgary/16_August_2009 16]
[http://2009.igem.org/Team:Calgary/17_August_2009 17] [http://2009.igem.org/Team:Calgary/18_August_2009 18] [http://2009.igem.org/Team:Calgary/19_August_2009 19] [http://2009.igem.org/Team:Calgary/20_August_2009 20] [http://2009.igem.org/Team:Calgary/21_August_2009 21] [http://2009.igem.org/Team:Calgary/22_August_2009 22] [http://2009.igem.org/Team:Calgary/23_August_2009 23]
[http://2009.igem.org/Team:Calgary/24_August_2009 24] [http://2009.igem.org/Team:Calgary/25_August_2009 25] [http://2009.igem.org/Team:Calgary/26_August_2009 26] [http://2009.igem.org/Team:Calgary/27_August_2009 27] [http://2009.igem.org/Team:Calgary/28_August_2009 28] [http://2009.igem.org/Team:Calgary/29_August_2009 29] [http://2009.igem.org/Team:Calgary/30_August_2009 30]
[http://2009.igem.org/Team:Calgary/31_August_2009 31]


September
MTWTFSS
  [http://2009.igem.org/Team:Calgary/1_September_2009 1] [http://2009.igem.org/Team:Calgary/2_September_2009 2] [http://2009.igem.org/Team:Calgary/3_September_2009 3] [http://2009.igem.org/Team:Calgary/4_September_2009 4] [http://2009.igem.org/Team:Calgary/5_September_2009 5] [http://2009.igem.org/Team:Calgary/6_September_2009 6]
[http://2009.igem.org/Team:Calgary/7_September_2009 7] [http://2009.igem.org/Team:Calgary/8_September_2009 8] [http://2009.igem.org/Team:Calgary/9_September_2009 9] [http://2009.igem.org/Team:Calgary/10_September_2009 10] [http://2009.igem.org/Team:Calgary/11_September_2009 11] [http://2009.igem.org/Team:Calgary/12_September_2009 12] [http://2009.igem.org/Team:Calgary/13_September_2009 13]
[http://2009.igem.org/Team:Calgary/14_September_2009 14] [http://2009.igem.org/Team:Calgary/15_September_2009 15] [http://2009.igem.org/Team:Calgary/16_September_2009 16] [http://2009.igem.org/Team:Calgary/17_September_2009 17] [http://2009.igem.org/Team:Calgary/18_September_2009 18] [http://2009.igem.org/Team:Calgary/19_September_2009 19] [http://2009.igem.org/Team:Calgary/20_September_2009 20]
[http://2009.igem.org/Team:Calgary/21_September_2009 21] [http://2009.igem.org/Team:Calgary/22_September_2009 22] [http://2009.igem.org/Team:Calgary/23_September_2009 23] [http://2009.igem.org/Team:Calgary/24_September_2009 24] [http://2009.igem.org/Team:Calgary/25_September_2009 25] [http://2009.igem.org/Team:Calgary/26_September_2009 26] [http://2009.igem.org/Team:Calgary/27_September_2009 27]
[http://2009.igem.org/Team:Calgary/28_September_2009 28] [http://2009.igem.org/Team:Calgary/29_September_2009 29] [http://2009.igem.org/Team:Calgary/30_September_2009 30]


October
MTWTFSS
      [http://2009.igem.org/Team:Calgary/1_October_2009 1] [http://2009.igem.org/Team:Calgary/2_October_2009 2] [http://2009.igem.org/Team:Calgary/3_October_2009 3] [http://2009.igem.org/Team:Calgary/4_October_2009 4]
[http://2009.igem.org/Team:Calgary/5_October_2009 5] [http://2009.igem.org/Team:Calgary/6_October_2009 6] [http://2009.igem.org/Team:Calgary/7_October_2009 7] [http://2009.igem.org/Team:Calgary/8_October_2009 8] [http://2009.igem.org/Team:Calgary/9_October_2009 9] [http://2009.igem.org/Team:Calgary/10_October_2009 10] [http://2009.igem.org/Team:Calgary/11_October_2009 11]
[http://2009.igem.org/Team:Calgary/12_October_2009 12] [http://2009.igem.org/Team:Calgary/13_October_2009 13] [http://2009.igem.org/Team:Calgary/14_October_2009 14] [http://2009.igem.org/Team:Calgary/15_October_2009 15] [http://2009.igem.org/Team:Calgary/16_October_2009 16] [http://2009.igem.org/Team:Calgary/17_October_2009 17] [http://2009.igem.org/Team:Calgary/18_October_2009 18]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/19_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/20_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/21_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/22_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/23_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/24_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/25_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 25]
[http://2009.igem.org/wiki/index.php?title=Team:Calgary/26_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/27_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/28_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/29_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/30_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Calgary/31_October_2009&preload=Team:Calgary/NotebookPreload&action=edit 31]



JULY 27, 2009


CAROL

Finishing Modeling Paper

  • Finished writing modeling paper that summarizes what we will be doing once the circuit is completed. The experiments should not take long to accomplished and these results can be tabulated in Matlab quickly.
  • Plated the week-long ligation of the promoter library in both XL Gold Ultra competent cells and Top 10 cells, but there is no growth.
  • A reason that we thought of that might have caused minimal colonies is the phosphatase step. The 32 primers that we have synthesized does not have 5' phosphates. If we phosphatase the vector, that might result in no ligation.
  • Re-started restriction enzyme digestion on vector pCS26 with XhoI and BamHI at 37oC for 4 hours. Then I ligated the promoter with the vector with Quick Ligase for 5 minutes. Then I transformed the plasmids into both XL Gold Ultracompetent cells and Top 10 cells. Incubate at 37oC for 16 hours.


CHINMOYEE

Descriptive Title of What You're Doing

WIKI CODING HERE


EMILY

Descriptive Title of What You're Doing

WIKI CODING HERE


FAHD

Descriptive Title of What You're Doing

WIKI CODING HERE


IMAN

Descriptive Title of What You're Doing

WIKI CODING HERE


JAMIE

Descriptive Title of What You're Doing

WIKI CODING HERE


JEREMY

Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers

Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers
Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.

Protocol
  MM1 (6x) (μL) MM2 (6x) (μL)
10X PCR buffer minus MgCl2 30 30
10mM dNTPs 6 6
50mM MgCl2 9 9
F primer 6 (BBK CP F) 6 (LuxPQ F)
R primer 6 (BBK CP R) 6 (LuxOU R)
ddH2O 241.8 241.8
pTaq 1.2 1.2


Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3

PCR conditions

# of cycles Temp (ºC) Time
1 94 6 min
36 94 30sec
55 45sec
72 6min 20sec
1 72 10min
  4 hold

Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.




Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers
Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)
Protocol

  MM1 (8x) (μL) MM2 (6x) (μL)
10X PCR buffer minus MgCl2 40 40
10mM dNTPs 8 8
50mM MgCl2 12 12
F primer 8 (LuxPQ F) 8 (BBK CP F)
R primer 8 (LuxPQ R) 8 (BBK CP R)
ddH2O 322.4 322.4
pTaq 1.6 1.6

Positive control = ∆LuxPQ in psB1AK3

PCR conditions
<hmtl>

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# of cycles Temp (ºC) Time
1 94 6 min
36 94 30sec
55 45sec
72 6min 20sec
1 72 10min
  4 hold
pTaq 1.6 1.6

</html>

Image


Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)
Purpose: isolate and measure concentrations of pure plasmids.

DNA 260/280 260/230 Conc. [ng/μL]
∆LuxPQ in AC3 C1 1.83 2.23 146.8
∆LuxPQ in AC3 C2 1.81 2.19 112.6
∆LuxPQ in AC3 C3 1.8 2.27 145.8
∆LuxPQ in AC3 C4 1.78 2.09 96.6
∆LuxPQ in AC3 C5 1.81 2.25 115.3
∆LuxPQ in AC3 C6 1.79 2.17 44.8
PQ-B-R-OU-B in AC3 C1 1.76 1.86 29.4
PQ-B-R-OU-B in AC3 C2 1.59 1.44 20.1
PQ-B-R-OU-B in AC3 C3 1.7 2.27 16.3
PQ-B-R-OU-B in AC3 C4 1.71 1.79 17




Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
Protocol
Recipient

Recipient 1 Recipient 2
2μL REact 4 2μL REact 4
0.75 μL SpeI 0.75 μL SpeI
0.75 μL PstI 0.75 μL PstI
2 μL of ∆LuxPQ in AK3 C2 [112.6ng/μL] 2 μL of ∆LuxPQ in AK3 C5 [115.3ng/μL]
14.5 μL ddH2O 14.5 μL ddH2O


Insert
2μL REact 2
0.75 μL XbaI
0.75 μL PstI
2 μL of LuxPQ-B0015-R0040-LuxOU-B0015 in AK3 C1 [156.3ng/μL]
12.5 μL ddH2O


Put in the incubator at 37ºC overnight.



KATIE

Descriptive Title of What You're Doing

WIKI CODING HERE


KEVIN

Descriptive of What You're Doing

WIKI CODING HERE


MANDY

Descriptive Title of What You're Doing

WIKI CODING HERE


PATRICK

Descriptive Title of What You're Doing

WIKI CODING HERE


PRIMA

Descriptive Title of What You're Doing

WIKI CODING HERE


STEFAN

Descriptive Title of What You're Doing

WIKI CODING HERE


VICKI

Descriptive Title of What You're Doing

WIKI CODING HERE

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